Infectious diseases and natural defence
Man is constantly at risk for chronic injections by a variety of agents such as viruses, bacteria, fungi, protozoa and multicellular parasites. Some of the associated infectious diseases are seriously disabilitating or even life-threatening. In response to the threat exerted by these pathogens a variety of defence mechanisms, the so called immune responses, have evolved in mammalians. For an excellent overview on the topic see chapters 1, 2, 15 and 16 in ref.1.
A distinction can be made between innate (or non-adaptive) immune responses and adaptive immune responses. The latter type of response is highly specific for a particular pathogen and improves with each successive encounter with the infectious agent. The adaptive immune responses are mediated by various types of lymphocytes. The innate immune responses are primarily produced by the phagocytic cells. These more primitive responses are not based on a highly specific recognition and act as a first line of defence against infection. An important group of phagocytes are long-lived cells (monocyte/macrophages) that belong to the mononuclear phagocyte lineage. Monocytes are formed from bone marrow stem cells and enter the blood stream. These cells can migrate out into the tissue where they develop into various types of tissue macrophages. Examples are the microglial cells in brain, the alvcolar macrophages in the lung, the Kupffer cells in the liver, the mesanglial phagocytes in the kidney, the splenic macrophages, the lymph node resident and recirculating macrophages and the synovial cells. The second category of phagocytes is formed by the polymorphonuclear neutrophils that are short-lived cells and constitute the majority of the blood leukocytes.
Role of phagocytes in defence
Well documented is the key role played by phagocytes in immunity to bacterial infections. Phagocytes are attracted chemotactically to a bacterial infection. Attachment to the bacterium can occur via numerous interactions, e.g. complement-mediated, antibody-mediated or mannose-binding protein-mediated or via lectin-oligosaccharide interaction. Subsequently the organism is exposed to a sequence of killing mechanisms in phagosomes and lysosomes. Of great importance are the oxygen-dependent killing mechanism that generate the superoxide anion and subsequently other reactive oxygen intermediates that are toxic. More recently the importance of killing via the nitric oxide pathway in neutrophils has become evident. Oxygen-independent killing is mediated by defensins, small cationic polypeptides, lysosomal enzymes, lysozyme and lactoferrin. The precise roles of phagocytes in immunity to fungal and parasite infections are less well understood, but it is thought that they are similar to those involved in resistance to bacterial infections.
Besides their direct role in killing of organisms, macrophages play other important roles in the immunity to foreign organisms. Firstly, these cells are very effective at presenting antigens to T lymphocytes followed by further responses of the immune system. Secondly, exposure of macrophages to microbial products can be accompanied by release of cytokines that affect other components of the immune system. Thirdly, macrophages respond to cytokines released by T lymphocytes, for example, in some parasite infections the body reduces damage by walling off the parasite behind a capsule of inflammatory cells. The T-lymphocyte dependent process results in local accumulation of macrophages that release fibrogenic factors which stimulate the formation of granulomatous tissue and ultimately fibrosis.
Intervention of infectious diseases
The natural defence mechanisms against pathogens are not always sufficiently effective to prevent clinical complications and (preventive) intervention is therefore required.
One preventive approach is immunization, i.e. stimulation of defence mechanism by prior vaccination of the host with (components of) pathogens. To be effective, a vaccine must induce a long-lived response from the right king of T-lymphocytes that produce a strong cell-mediated immunity. Although vaccination has proved to be effective for some pathogens, a number of intrinsic problems are associated with this approach. Most importantly, it has to be avoided that antigens used for vaccination induce the wrong kind of immune response, such as suppression or even autoimmunity. With some infections there is a need to achieve immunity in specific body locations that can be only obtained by local or oral immunization. Due to the complexity of the immune system, the heterogeneity in pathogens and the ability of some pathogens to escape from the specific immune responses, a generalized approach for effective immunization against pathogens is not available. Various strategics for specific infectious diseases remain under investigation by trial and error.
Another approach in the intervention of infectious diseases is the use of pharmacological agents that prevent further proliferation or survival of pathogens. In this connection use is made preferably of compounds that specifically act at the level of the pathogen and do not affect the host. Such specificity is based on differences in the composition and needs of mammalian cells and their pathogenic invaders. Some illustrative examples are the following. Penicillins and cephalosporins are specific inhibitors of bacterial cell wall synthesis. Aminoglycosides, chloramphenicol, tetracyclines, macrolides are inhibitors of bacterial protein synthesis; rifampicin, 4-quinolones are specific inhibitors of bacterial DNA replication. Amphotericin B and nystatin are antibiotics that are fungicidal due to binding specific sterols in the fungal cell membrane, thus causing leakage of cell components.
The pharmacological approach to intervene with pathogens at the level of a specific target that is absent i the host cells is also used in nature. A good example is the hydrolase lysozyme that is present in vertebrates as well as many invertebrates. Cell walls of many bacteria contain interlinked polymers of muramic acid and N-acetylglucosamine. The hydrolase lysozyme is capable of cleaving the glucosidic bond between muramic acid and N-acetylglucosamine moieties and consequently the integrity of the cell wall. The presence of lysozyme is without harm for the host since a similar structure is absent in non-bacterial cells.
Chitin
Chitin is a glycopolymer that is absent in mammalian cells but is present in a variety of organisms that cause infectious diseases in man. Chitin therefore forms an attractive target for selectively attacking these type of pathogens.
Chitin is a polymer of .beta.(1-4) linked N-acetyl-D-glucosamine units. It may also contain glucosamine units in different proportions. Mainly deacetylated chitin is call chitosan. For overviews on the topic see refs. 2-5.
Chitin and its derivatives are one of the most abundant macromolecular biological products on earth. The estimated annual production is 10,000-100,000 million tons. Chitin is a structural component of cell walls of fungi and of the exoskeleton of almost all invertebrates (except sponges, most anthozoa, scyphozoa, and echinoderms), but is absent in vertebrates and autotrophic organisms. Chitin fulfils important functions: it protects cells and organisms against mechanical and chemical stress from the environment and it also supports and determines their shape. The chain length of N-acetylglucosamine polymers may differ from 100 to 8000 units. The polymers assemble laterally to form microfibrils, stabilized by strong hydrogen bonds between the amine group of sugar in one chain and the carbonyl group of sugar in a neighbouring chain. Three crystallographic forms of chitin can be recognized. In .alpha.-chitin, the most abundant form in fungi and arthropods, adjacent chains are oriented antiparallel. In .beta.-chitin the chains are oriented parallel, whilst in .gamma.-chitin two chains are parallel and the third one anti-parallel. The microfibrils in crustacea and fungi usually show a diameter of 20-25 nm. In most structures, chitin is associated with other substances. In fungal cell walls the accompanying compound is .beta.-glucan. In exoskeletons of animals, chitin protein associations are however predominant. The matrix is hardened by deposition of calcium carbonate and phosphate as in crustacea or by tanning with phenolic derivatives as in insects. In chitinous structures of protozoa also glycoproteins and mucopolysacharides are present.
Chitin and related compounds have found many applications. Chitosan is used as component of threads, fibers, films and gels. In the agricultural industry, seeds can be protected from fungi using a capsule containing chitin derivatives. In the food industry, chitosan is used in the preparation of fruit juices and soluble coffee. The cosmetic industry produces shampoos, gels, creams and even sponges containing chitosan. In the pharmaceutical industry and in medicine, chitosan occurs in the making of contact lenses, of drug excipients and of dressings for burns.
Chitin synthesis and degradation by chitinases
The synthesis of chitin is the best understood for fungi. The essential precursor is UDP-N-acetylglucosamine that is synthesized from glucose. Chitin synthetases are transported as transmembrane proteins to the plasma membrane where they add N-acetylglucosamine from the donor UDP-N-acetylgluosamine across the membrane to the growing polysaccharide chain. Fungal synthetase can be competitively inhibited by polyoxins (produced by Streptomyces cacaol) and nikkomycins (produced by Streptomyces tentae), both being analogues of UDP-N-acetylglucosamine.
Chitin synthesis in other species is less well documented. It is suggested that in insects and crustacea chitin synthesis begins in the edoplasmic reticulum via glycosylation of a protein to which a chitin chain is added in the Golgi apparatus. The chitoprotein is subsequently exported to the cell surface. Chitin synthesis is arthropods appears to be a two-step process involving lipid-linked intermediates. The synthesis in arthropods can be specifically inhibited by insecticides of the benzoylphenylurea type of which the mechanism of action is still not precisely known. Furthermore inhibitors of protein synthesis and N-linked glycosylation (tunicamycin) inhibit the synthesis of chitin in these organisms. Evidence so far suggests that chitin synthesis in protozoa occurs at the cell surface and most likely resembles the process in fungi.
All chitin-containing organisms presumably contain enzyme systems that allow them to degrade the chitin polymer in order to allow morphogenesis, i.e. essential modifications of their shape. Furthermore, many higher plants, fish and insectivorous animals (including vertebrates) are capable of producing enzymes that can degrade chitin. Several enzymatic systems are in this respect distinguisable: i) .beta.-hexosaminidases are capable of removing the terminal N-acetylglucosamine moiety from the non-reducing end of the polysaccharide; ii) some lysozymes with a broad specificity (e.g. egg white lysozyme) are capable to cleave also within the chitin glycopolymer; iii) so called exochitinases cleave diacetylchiobiose units from the non-reducing end of the polysaccharide; and iv) specific endochitinases cleave glycosidic linkages randomly along the chitin chain, eventually giving diacetylchitobiose as major product, together with some triacetylchitotriose. The exo- and endochitinases are often exclusively named thitanases. This nomenclature is also used herein.
Chitinases are widespread in nature and have been found in some viruses, bacteria, fungi, plants, invertebrates and vertebrates. Chitinases constitute families 18 and 19 of glycosylhydrolases. This classification proposed by Henrissat is based on amino acid sequence similarity (6). Family 19 only contains plant chitinases; for example, the chitinase from Hordeum vulgare for which the 3-dimensional structure has been resolved. Only the so called class III plant chitinases belong to the family 18 of glycosylhydrolases. There is a considerable homology in the putative active site regions in chitinases of the family 18 of glycosyl-hydrolases. The proposed structure for the catalytic domain is a 8-stranded .alpha./.beta. barrel (`TIM barrel`) (7,8). The reaction mechanism seems to be similar to that of lysozyme and most other glycosylhydrolases, i.e. general acid-base catalysis (8).
Infectious diseases in man caused by pathogens containing chitin
A variety of infectious diseases in man are caused by organisms that contain chitin. The most prominent ones are listed in Table 1. On the basis of the type of pathogen a classification can be made in: i) fungal infections; ii) protozoal infections, and iii) helminth (worm) infectious. For an overview on the topic see for example ref.9.
TABLE 1 ______________________________________ Some infectious diseases caused by chitin-containing pathogens ______________________________________ I. Fungal infections Cutaneous mycoces Subcutaneous mycoces Pulmonary mycoces Candidiasis II. Protozoal infections Toxoplasmosis Malaria (Plasmodium species) Leishmaniasis (Leishmanic species) Chagas disease, sleeping sickness (Tryponosoma species) III. Helminth infections Schistosomiasis Trichinosis Filariasis Ochocerciasis ______________________________________
Fungal infections
The limited number of presently available anti-fungal drugs are in general not very potent. Fungal infections are regularly encountered in immune-incompetent people, currently most frequently in patients with acquired immunodeficiency syndrome (AIDS). Most fungal infections of the skin are treated with topical preparations. Visceral infections and cuticular infections require prolonged systemic therapy.
The most frequent fungal infection is caused by Candida albicans. The organism is a common commensal of the oral and vaginal mucosae but can become a pathogen on damaged skin, in severely ill patients, in patients who have specific immune deficiency, and in patients receiving broad-spectrum antibiotics when the local microbiol ecology is disturbed. Extreme consequences of Candida infection can be pneumonia, endocarditis, septicaemia and even death. The only effective treatment is intravenous administration of amphotericin B. Administration of this drug can result in serious adverse affects that are accompanied by hypotension and collapse. For that reason an initial test dose is infused to determine tolerance. Flucytosine is a synthetic fluorinated pyrimidine which enters fungal cells and inhibits metabolism by interfering with DNA and RNA synthesis. The compound is usually given in combination with amphotericin B for treatment of systemic fungal infections. When administered alone, resistance towards flucytosine rapidly develops.
Other species of fungi that can cause severe infectious diseases in man are Aspergillus, Cryptococcus, Coccidinioides, Paracoccidioides, Blastomyces, Sporothrix, and Histoplams capsulatum.
The clincal features of the more commonly encountered histoplasmesis may differ considerably. Histoplasma capsulatum infects macrophages and the pathogenesis of the disease is in some aspects similar to that of tuberculosis. In normal hosts acute pulmonary infection is often accompanied by cough and chest pain, myalgia and weight loss. In individuals with structural defects of the lung a chronic destructive disease in the lung apices may develop, similar to tuberculosis. In immunocomprised hosts disseminated histoplasmosis may develop, accompanied by fever, hepatosplenomogaly, anaemia, leucopenia, thrombocytopenia and pneumonia. Amphotericin B is the common choice of treatment. In view of its toxicity, treatment of various fungal infections with other drugs is investigated.
Protozoal and helminth infections
Protozoa are single-cell organisms that are causing a large number of severe infectious diseases in man (see Table 1). Fortunately for most of these pathogens effective treatment with drugs is feasible. The treatment of Chagas disease, caused by Trypanosoma cruzi, is at present not satisfactory. The heart and the gut are the organs severely affected in the chronic form of this disease. Effective treatment of tissue cysts of Toxoplasma gondii is not feasible. Reactivation of the disease may occur following depression of cell-mediated immunity. Helminth infections can be generally quite effectively cured with specific drugs. A major health problem in this respect is formed by lymphatic filariasis caused by Wucheria bancrofti and Brugia species. In an advanced state of the disease killing of microfilariae by the drug diethylcarbamzine (DEC) may result in complications as the result of responses to death of massive amounts of worms.
Improved resistance against chitin-containing pathogens by interaction at the level of chitin
The differential distribution of chitin among organisms has lead to the idea that chitin metabolism is an attractive target for controlling infections by chitin-containing organisms. Two distinct experimental approaches should be mentioned.
1. Chitin synthesis inhibition (for a review see ref.2)
The value of inhibitors of fungal cell wall synthesis as fungicides has been largely investigated in plants. Polyoxins have been widely used as excellent agricultural fungicides. The polyoxins are a group of related competitive inhibitors of the chitin synthetase reaction due to their structural resemblance to UDP-N-acetylglucosamine. More recently, nikkomycin has been detected as a potent inhibitor with a similar mechanism of action.
Benzoylaryl ureas are commercial insecticides which are highly potent inhibitors of chitin synthesis in insects but not in fungi.
Medical applications in man of the above compounds have not been documented.
2. Chitinases as vaccine
The importance of chitinase activity in the life cycle of protozoa has stimulated several investigators to consider the protist chitinases an attractive antigen for vaccination (10-13). Moreover, because it is so far (incorrectly) assumed that analogous proteins are not present in man (see e.g. ref.13).
The experimental approaches described above may be less advantageous in the battle against chitin-containing organisms than assumed.
Firstly, inhibition of chitin synthesis by synthetic compounds may be less specific and effective than hoped for. It cannot be excluded that inhibitors also interfere with endogenous processes in mammalian cells and consequently result in side-effects. Toxicity could also arise as a result of biotransformation of the original compound to a toxic product. Furthermore, prolonged administration of large quantities of drugs may be required due to the fact that these compounds are excreted via the urine. Moreover, alternative synthesis routes of chitin may exist or develop in organisms, complicating its complete inhibition.
Secondly, the use of chitinases from pathogenic organisms as a vaccine may result in unforeseen harmful side effects. It cannot be excluded that fragments of such chitinases share homology with endogenous proteins and that an undesired immune response is elicited. This may in fact be more than a theoretical problem because of the strong homology between human chitinase and chitinases from other species (see below).
Role of chitinases in plants and fish in resistance against chitin-containing pathogens
Many plant chitinases are considered pathogenesis-related (PR) proteins. The enzymes are induced by the presence of (extracts of) pathogens, or other forms of stress. For some of the plant chitinases an anti-fungal role has been documented in vitro. For an overview see ref.14. Most strikingly, it was reported that spraying plants with a bacterial chitinase (ChiA) from Serratia marcesens expressed in E. coli renders protection against fungi (15). It is clear however that more effective inhibition of most fungi requires the concomitant presence of chitinase and .beta.-1,3-glucanases. In plants, the latter enzymes are also induced in response to stress.
It has been reported that leucocytes of fish are rich in chitinase activity and fulfil a role in defence (16). Evidence for such a role was recently provided by the demonstration of an inhibitory action of purified chitinase from turbet against the chitinous fungus Mucor mucedo.
At present it is generally believed that man does not contain a comparable chitinase in phagocytes. However, as will be discussed in detail below, we noted recently the presence of a similar type of enzyme in cultured human macrophages.